Bacterial meningitis is an inflammatory disease of the central nervous system caused by the growth of bacteria in and adjacent to the leptomeninges. It is an acute, and often lethal, infectious disease that affects children and young adults. One of the most common causes of bacterial meningitis worldwide is infection with Neisseria meningitidis. The occurrence of infection with this bacterium is unpredictable, since many people become colonized without exhibiting the disease. Some people are temporary carriers, while others are chronic carriers, discharging meningococci either more or less continuously or in a sporadic fashion. During the course of infection, bactericidal antibodies are produced in the infected person, which apparently immunize the person against subsequent infection (Goldschneider, I., et al., J. Exp. Med. 129:1327-1348 (1969)). This observation has led to the expectation that vaccines based on bacterial antigens may be effective against meningitis.
N. meningitidis is a Gram-negative coccus. Characteristically, it is surrounded by a cell envelope composed of an inner plasma membrane, a periplasmic space, and an outer membrane or cell wall. The outer membrane is composed of lipooligosaccharide (LOS) molecules, lipids, proteins and polysaccharides. The protective antibodies produced in infected people were found to be directed against both the capsular polysaccharides and the outer membrane proteins (Frasch, C. E., In Medical Microbiology (eds. C. S. F. Easman and J. Jeljaszewicz), Academic Press, New York, Vol. 2, pp. 115-144 (1983)).
Strains of N. meningitidis have been classified into serogroups according to the type (antigenically and biochemically) of the capsule. Currently recognized serogroups include A, B, C, D, W135, X, Y, Z and 29E. The polysaccharides responsible for the serogroup specificity of groups A, B, C, X, W135 and Y have been purified. A tetravalent vaccine based on purified capsular polysaccharides from serogroups A, C, Y and W 135 has been developed (Hankins, W. A., et al., Proc. Soc. Exp. Biol. Med. 169:54-57 (1982)). However, the lack of immunogenicity in the under 2 years age group, the age group most at risk from meningococcal infection, has limited the usefulness of this vaccine. The capsule of Group B N. meningitidis is poorly immunogenic in all age groups, even when conjugated to a carrier protein. There is evidence that antibodies to this capsule may cross react with brain tissue of fetal and newborn infants.
The major outer membrane proteins (omp) of N. meningitidis have been divided into five classes on the basis of structural similarities, as determined by migration (Mr) on SDS-polyacrylamide gels and peptide map analysis (Tsai, C. M., et al., J. Bacteriol. 146:69-78 (1981)). Of these protein classes, the class 1 protein appears to be the most interesting for vaccine production. This antigen seems to be a major immunodeterminant in humans. It is expressed in most isolates of N. meningitidis and is the basis for subtype specificity of strains.
Several attempts have been made to produce a vaccine based on outer membrane proteins. Vaccines composed of capsular polysaccharide and outer membrane proteins, or just outer membrane proteins in a vesicular complex, have been tested. Only one of these vaccines has been reported to be more than 57% effective (Sierra, G. V. G., et al., NIPH Annals 14(2):195-210 (1991)).
Another significant problem in the development of both outer membrane and capsule based vaccines is the presence of the bacterial lipooligosaccharide (LOS), which produces toxic side effects in humans. LOS is also referred to as bacterial endotoxin. Low amounts of LOS can cause fevers, and high doses of LOS can result in a general wasting (cachexis) of the patient. The most recent outer membrane complex vaccines have had residual LOS levels of 10-70 xcexcg/mg protein (see Zollinger, W. D., In New Generation Vaccines (eds. G. C. Woodrow and M. M. Levine), Marcel Dekker, Inc., New York, pp. 325-348 (1990)). Thus, there exists a need for safe and effective vaccines for bacterial meningitis caused by N. meningitidis, and especially for disease caused by Group B strains.
This invention relates to a method for the effective removal of lipooligosaccharide (LOS) from outer membranes of Gram-negative cocci by sequential extractions with certain detergents. LOS, which is also referred to as bacterial endotoxin, can cause undesirable side effects in vaccines, such as fever. As described herein, the method produces outer membranes and soluble outer membrane proteins with an extremely low content of LOS, but which retain immunogenicity. These LOS-depleted outer membrane products are further shown to elicit bactericidal antibodies which confer protection against Neisseria meningitidis in animals. Thus, vaccines comprising LOS-depleted outer membranes and soluble outer membrane proteins are provided, which are expected to be useful in therapy and prophylaxis against meningitis and other diseases caused by Gram-negative cocci.
Specifically described herein are outer membrane products of Neisseria meningitidis. Outer membrane products prepared from N. meningitidis by the present method are shown to have a LOS content of less than about 0.01% (wt./wt. total protein) and to elicit antibodies which are both bactericidal and protective in animals. As a result, vaccines against neisserial meningitis are provided, which are immunogenically effective and relatively free of bacterial LOS. Of particular interest is a vaccine against serogroup B strains of N. meningitidis, for which there is presently no effective vaccine.
The present method is expected to also be applicable to other Gram-negative cocci, since these bacteria have structurally similar outer membranes. For example, LOS-depleted outer membranes of other Neisseria species, such as N. gonorrhoeae, and other Gram-negative cocci, such as Moraxella, can be prepared. Thus, N. meningitidis is representative of other Gram-negative cocci in the procedures and products described below.
This method is further applicable to various natural strains of Gram-negative cocci, as well as to recombinant strains that are genetically engineered to produce more than one subtype-specific epitope, e.g., more than one class 1 protein of N. meningitidis. Multivalent vaccines can be prepared using mixtures of strains or recombinant strains expressing the surface epitopes of several serogroups, serotypes or subtypes.
The method comprises sequential extractions with different kinds of detergents. First, total membranes of the cocci are extracted with a polyoxyethylene detergent (e.g., TRITON X-100(trademark), BRIJ35(trademark), or TWEEN80(trademark)), resulting in outer membranes that are depleted of inner membranes and some of the LOS. This is followed by extraction of the outer membranes with a zwitterionic betaine detergent, such as one of the ZWITTERGENT(trademark) series (e.g., 3-12 or 3-14) or EMPIGEN BB(trademark). These detergents specifically remove essentially all of the remaining LOS while extracting very little protein.
The resulting LOS-depleted outer membrane preparation is composed of outer membrane proteins (omps) complexed with cell wall components. This preparation can be used for vaccine purposes directly, or the omp can be solubilized and extracted from the other cell wall components using a zwitterionic betaine detergent in a salt buffer, e.g., with ZWITTERGENT 3-14(trademark) in about 0.1 to about 0.5 M NaCl. The solubilization step results in LOS-depleted fractions, one containing soluble outer membrane proteins and another containing insoluble outer membrane proteins complexed with other cell wall components. Both LOS-depleted outer membranes and LOS-depleted soluble outer membrane proteins are shown to elicit bactericidal antibodies in mice.